3‐Alkyl‐2‐Methoxypyrazines: Overview of Their Occurrence, Biosynthesis and Distribution in Edible Plants

Pyrazines are ubiquitous in nature – biosynthesized by microorganisms, insects, and plants. Due to their great structural diversity, they own manifold biological functions. Alkyl‐ and alkoxypyrazines for instance play a key role as semiochemicals, but also as important aroma compounds in foods. Especially 3‐alkyl‐2‐methoxypyrazines (MPs) have been of great research interest. MPs are associated with green and earthy attributes. They are responsible for the distinctive aroma properties of numerous vegetables. Moreover, they have a strong influence on the aroma of wines, in which they are primarily grape‐derived. Over the years various methods have been developed and implemented to analyse the distribution of MPs in plants. In addition, the biosynthetic pathway of MPs has always been of particular interest. Different pathways and precursors have been proposed and controversially discussed in the literature. While the identification of genes encoding O‐methyltransferases gave important insights into the last step of MP‐biosynthesis, earlier biosynthetic steps and precursors remained unknown. It was not until 2022 that in vivo feeding experiments with stable isotope labeled compounds revealed l‐leucine and l‐serine as important precursors for IBMP. This discovery gave evidence for a metabolic interface between the MP‐biosynthesis and photorespiration.

Pyrazines are ubiquitous in nature -biosynthesized by microorganisms, insects, and plants.Due to their great structural diversity, they own manifold biological functions.Alkyl-and alkoxypyrazines for instance play a key role as semiochemicals, but also as important aroma compounds in foods.Especially 3alkyl-2-methoxypyrazines (MPs) have been of great research interest.MPs are associated with green and earthy attributes.They are responsible for the distinctive aroma properties of numerous vegetables.Moreover, they have a strong influence on the aroma of wines, in which they are primarily grapederived.Over the years various methods have been developed and implemented to analyse the distribution of MPs in plants.
In addition, the biosynthetic pathway of MPs has always been of particular interest.Different pathways and precursors have been proposed and controversially discussed in the literature.While the identification of genes encoding O-methyltransferases gave important insights into the last step of MP-biosynthesis, earlier biosynthetic steps and precursors remained unknown.It was not until 2022 that in vivo feeding experiments with stable isotope labeled compounds revealed l-leucine and l-serine as important precursors for IBMP.This discovery gave evidence for a metabolic interface between the MP-biosynthesis and photorespiration.

-position.
As almost all N-heterocycles, pyrazine is a basic compound. [1]ver the years many synthetic routes to pyrazines have been developed.Amongst others, pyrazines are obtained by direct ring closure e. g. a cyclodimerization of α-aminoketones or αaminoaldehydes or a condensation of a 1,2-diketone with a 1,2diamine. [2]yrazines exhibit a wide range of biological functions like antibacterial, antiviral, and anticancer properties.That is why they are often used as pharmaceuticals.Pyrazinamide (1) as well as morinamide (2) are used for the treatment of tuberculosis, whereas oltipraz (3) serves as an anti-tumor agent (Figure 1). [3]Moreover, pyrazines are used as pesticides.Thionazin (4) for example serves as a nematicide. [4]owever, pyrazines are not only synthesized chemically, but occur also in nature, where they are widespread.][9] Aspergillic acid, which has antibiotic and antimycotic properties for example is a commonly known pyrazine biosynthesized by Aspergillus flavus. [10]n nature, pyrazines are often used as semiochemicals serving as pheromones for intraspecific communication or as allelomones for interspecific communication. [8,11,12]Four semiochemically active pyrazines are displayed in Figure 2. 3-Ethyl-2,5-dimethylpyrazine ( 5) is biosynthesized by an ant-associated bacterium (Serratia marcescens) and serves as an important trail and alarm pheromone of ants (Atta sexdens rubropilosa). [13,14],5-Dimethyl-3-(2-methylbutyl)-pyrazine (6) is used for the chemical defense by the phasmid insect Phyllium westwoodii.[15] Several trisubstituted alkylpyrazines like 2-propyl-3,5-dimeth-ylpyrazine (7) are used for the pollinator attraction of Drakaea glyptodon flowers.[16] 3-Isopropyl-2-methoxypyrazine (IPMP) (8)  turned out to be biosynthesized as an attractant by ladybird beetles, but is also known to be emitted by the roots of spinach (Spinacea oleracea) to attract the root knot nematode Meloidogyne incognita.[17] In addition, pyrazines serve as key odorants in foods.Due to their flavor and aroma properties, they are frequently used for improving the organoleptic quality of food products.[18] A compound's sensory impact on the overall aroma of food is defined as its odor activity value (OAV).The OAV is calculated as the ratio of a compound's concentration in the sample to its odor detection threshold (OT), which in turn is defined as the lowest concentration that can still be perceived by the human sense of smell. [19]specially, alkyl-and alkoxypyrazines possess low OT, as they are extremely odorous even when present in low amounts.[18,[20][21][22] It was shown in 2021 that the pyrazine-selective olfactory receptor (OR) called OR5 K1 is responsible for humans perceiving the odor of pyrazines.Notably, OR5K1 is especially activated by pyrazines, which serve as semiochemicals and key aroma compounds. Thei perception is assumed to depend on the receiver: 2,3,5-trimethylpyrazine (Figure 3, 8) for example is perceived as aversive by mice, for which it serves as an alarm  semiochemical, while it is attractive to humans due to its roasted nut-or cocoa-like smell.Unnatural processes like roasting are thought to be responsible for this ambiguous perception.[23] 2. Alkylpyrazines

Occurrence
Alkylpyrazines are known to contribute to the roasted or nutty flavour of foods. [27]They occur in a wide range of heat-treated foods like baked bread, roasted peanuts, or coffee powder as well as in fermented products like miso, natto, and soy sauce. [20,28]As it is known, pyrazines are formed during thermal food processing.As typical Maillard reaction products they are generated by a condensation of a reducing carbohydrate with an amino group. [29]However, an alternative pathway via azomethine ylides (14), which are formed by a condensation of α-keto acids and α-amino acids, was proposed in 2010 (Scheme 1). [30,31]

Biosynthesis
Furthermore, alkylpyrazines can be biosynthesized by insects, bacteria, and plants. [7,12,32]Due to the great structural diversity of alkylated pyrazines, several biosynthetic pathways have been proposed, but not all have been elucidated yet.
Feeding experiments with Corynebacterium glutamicum revealed the biosynthesis of mono-and multi-alkylated pyrazines.Their biosynthesis starts with a non-enzymatic amination of acyloin (15), which generates α-aminocarbonyls (16).A selfcondensation of two α-aminocarbonyls yields a dihydropyrazine Francesca Zamolo received her master's degree in food chemistry from the University of Bonn in 2019 and was appointed a German state certified food chemist in 2020.The same year she started her doctoral studies under the supervision of Prof. Matthias Wüst at the University of Bonn.Her research is focused on the investigation of the biosynthetic pathway of 3-alkyl-2-methoxypyrazines in plants.
While sample clean-up was mainly conducted by solidphase extraction (SPE), extraction techniques varied. [37,38][45] In fact, the HS-SPME sampling technique was frequently used for the extraction of MPs from a wide range of matrices. [46]S-SPME is a rapid, non-exhaustive, and solvent-free sample preparation technique invented by Arthur and Pawliszyn in 1990. [47]It can be used for quantitative analysis providing constant and reproducible extraction conditions as well as appropriate calibration methods. [48]A highly precise quantification method is the stable isotope dilution assay (SIDA), which was introduced to aroma research notably by Schieberle and Grosch in 1987. [49]As the use of a stable isotope labeled compound leads to accurate results, SIDA is widely used for the quantification of MPs in different matrices like coffee beans, wine, and fragrant vegetable oils. [41,50,51]or MP-analysis, GC was hyphenated with different detection techniques like nitrogen phosphorus detection (NPD), flame ionization detection (FID), and mass spectrometry (MS). [42,52,53]GC turned out to be the main separation technique.Only a few studies used LC for the chromatographical separation of MPs. [54]Over the years, GC × GC-MS-hyphenation became increasingly important for the analysis of MPs in a highly complex matrix, as it provides a high peak capacity and thus enables the separation of a wide range of analytes. [53,55]

Occurrence
MPs occur in trace quantities in several foods including raw vegetables as well as raw peanuts, coffee, and fragrant vegetable oils. [9,36,50,56,57]The most common MPs are 3-isopropyl-2-methoxypyrazine (IPMP, 36), 3-sec-butyl-2-methoxypyrazine (sBMP, 37), and 3-isobutyl-2-methoxypyrazine (IBMP, 38) (Figure 8).IBMP was first identified in bell pepper fruits (Capsicum annuum L.) in 1969, followed by the identification of sBMP in galbanum oil (Ferula galbaniflua) and IPMP in green peas (Pisum sativum). [34,58,59]sually, all three MPs are present, but one is predominantly biosynthesized. [9,60]Beetroot (Beta vulgaris) and parsnip (Pastinaca sativa) for instance contain the highest sBMP-concentrations, while pea shells (Pisum sativum) are rich in IPMP. [9]he highest IBMP-levels can be found in unripe, green bell pepper fruits (Capsicum annuum var.grossum). [9]SIDA-HS-SPME-GC-MS-analysis demonstrated that IBMP is distributed within the whole bell pepper plant.The pericarp tissue of unripe bell pepper fruits turned out to be the main site of IBMP-storage.In general, IBMP-concentrations in the reproductive plant parts (flowers and fruits) exceeded those in the vegetative organs (roots, stems, leaves).Moreover, IBMP-levels of unripe fruit tissues were higher than those in ripe fruit tissues. [61]n addition, MPs are found in several grapes of Vitis vinifera varieties like Cabernet Sauvignon, Sauvignon Blanc, Cabernet Franc, Pinot noir, Riesling, Chardonnay, Merlot, and Carmenere, but also in grapes of Vitis species like V. amurensis, V. cinerea, V. riparia, and V. rupestris. [37,42,45,62,63]IBMP turned out to be the most abundant MP in grapes and wines.The IBMP-accumulation in bell pepper plants is comparable to the IBMPaccumulation in grapevines.In both cases, IBMP is distributed throughout the whole plant, but fruits are the main site of IBMP-accumulation. [60,64,65]t could be shown that IBMP-levels decrease during grape and bell pepper fruit ripening. [35,61]A degradation of IBMP (Scheme 5, 39) back to IBHP (40) has been supposed. [35]owever, decreasing IBMP-levels and increasing IBHP-levels did not correlate during bell pepper fruit ripening.In fact, IBHPlevels did not remain stable, but decreased during fruit maturation. [66]That is why a coexistence of further degradation pathways as well as a volatilization of IBMP during fruit maturation have been suggested, but have not yet been confirmed. [35,60]n addition, the reasons for IBMP-accumulation in the vegetative plant parts are still unknown.Different biological functions of MPs have been discussed in the literature.It has been suggested that MPs serve as semiochemicals.IPMP for  35). [26]gure 8.Chemical structures of 3-isopropyl-2-methoxypyrazine (IPMP, 36), 3-sec-butyl-2-methoxypyrazine (sBMP, 37), 3-isobutyl-2-methoxypyrazine (IBMP, 38).Scheme 5. Supposed degradation of IBMP (39) back to IBHP (40). [35]nstance has been shown to attract the knot nematode Meloidogyne incognita and IBMP and sBMP were found to be biosynthesized for chemical defense in aposematic insects like the wood tiger moths (Arctia plantaginis). [8,67]Since volatile organic compounds are often released by plants to interact with other organisms, it is proposed that IBMP acts as an alarm semiochemical to defend the unripe fruits from being eaten. [60,68]s grape-derived aroma compounds, MPs can also influence wine quality.MPs contribute to the typical characteristics of wine varieties such as Sauvignon Blanc in moderate concentrations, but can have detrimental effects when present in higher concentrations. [40,41,63]Estimated OT in wines are 10-16 ppt for IBMP and 1-2 ppt for IPMP. [34,45,58,63,69]While IBMP is mainly associated with a green bell pepper aroma, IPMP is primarily related to an earthy and herbaceous aroma. [70]An aroma associated with unripe and green attributes is perceived by the consumers at IBMP-levels � 8 ppt in white wine and � 15 ppt in red wine. [45,63]owever, MPs in wine are not only grape-derived.IPMP for example is considered to be the main cause for the off-flavor 'ladybug taint' (LBT), which is caused by the unintentional carryover of the Coccinellidae ladybeetles Harmonia axyridis and Coccinella septempunctata into the wine during winemaking. [71]oreover, cork stoppers can lead to elevated MP-levels in wine. [72]IBMP and IPMP were identified as corky off-flavor compounds that can migrate from cork stoppers into the wine. [73]76] Furthermore, MPs can have a significant impact on coffee quality.Elevated IPMP-and IBMP-levels are supposed to be responsible for the 'potato-taste defect' (PTD) found in roasted coffee made of Coffea species. [77]The PTD is associated with potato peel aroma. [78]It has been hypothesized that an insectbacteria-interaction between the coffee bug (Antestiopis orbitales) and bacteria species of the genus Enterobacteriaceae and Pantoea is causing an upregulation of genes and enzymes, which are involved in the biosynthesis of MPs. [79]

Biosynthesis
Over the years, different pathways of MP-biosynthesis have been proposed.The first step in MP-biosynthesis, which has been confirmed, was the enzymatically catalyzed O-methylation of 3-alkyl-2-hydroxypyrazines (HPs, 41) to MPs (42).In 2001, Sadenosyl-l-methionine (SAM) dependent enzymes (O-methyltransferases, OMTs) were identified as key enzymes in the biosynthesis of MPs. [80,81]][84] It has been shown that the VvOMTs differ in their affinities to catalyze the O-methylation of IBHP to IBMP (Scheme 6), which are caused by steric hindrance and thus larger distances between the methyl-acceptor (HPs) and methyl-donor (SAM). [60,85]n contrast, biosynthetic precursors and intermediates have long been unknown.Naturally occurring branched chain amino acids (BCAAs) such as l-leucine, l-isoleucine, and l-valine have usually been considered a potential precursor for MPs due to their similarity in the branched side chain. [9,58,75]n 1970, Murray et al. postulated a biosynthetic pathway for IPMP starting with the amination of valine (43) followed by a condensation with glyoxal (44) forming 3-isopropyl-2-hydroxypyrazine (IPHP, 45), which would in turn be enzymatically methylated to IPMP (46) (Scheme 7). [58]As neither α-amino acid amides nor free glyoxal had been detected in plants, this pathway has been discussed controversially. [9,86]ubsequently, an alternative pathway for the formation of IPMP has been proposed.Based on labeling studies in bacteria, it has been assumed that the biosynthesis of IPMP (50) starts with a condensation of valine (47) and glycine (48) yielding 3isopropylpiperazine-2,5-dione (49) (Scheme 8). [75,76]nother biosynthetic pathway was proposed in 2010.The addition of exogenous leucine and leucinamide (2-amino-4methylpentanamide, AMPA) to Cabernet Sauvignon grapes resulted in a significant increase in IBMP-content.Hence, it has Scheme 6. Final enzyme-catalyzed step in the biosynthesis of 3-alkyl-2methoxy-pyrazines (MPs).Methylation of 3-isobutyl-2-hydroxypyrazine (IBHP, 41) to 3-isobutyl-2-methoxypyrazine (IBMP, 42) in Vitis vinifera grapes catalyzed by S-adenosyl-l-methionine dependent Vitis vinifera O-methyltransferases (VvOMTs). [60,80,81,83]heme 7. Postulated biosynthesis of 3-isopropyl-2-methoxypyrazine (IPMP, 46) in plants. [58]heme 8. Postulated biosynthesis of 3-isopropyl-2-methoxypyrazine (IPMP, 50) in Pseudomonas species. [75]een hypothesized that leucine (51) serves as a precursor in the biosynthesis of IBMP (53), while AMPA (52) should act as an intermediate (Scheme 9). [87]he hypothesis of natural amino acids being precursors of MPs was strengthened by enantioselective sBMP-analysis, as only (S)-sBMP was detected in raw vegetables.As displayed in Scheme 10 the stereochemistry of l-isoleucine (53) is maintained during its incorporation into (S)-sBMP (54). [56]t was not until 2022 that stable isotope labeling experiments clearly proved that the biosynthesis of IBMP is linked to the amino acid metabolism. [88,89]In vivo feeding experiments combined with HS-SPME-GC × GC-ToF-MS-analysis turned out to be an efficient technique for tracking precursors of IBMP.Feeding studies were conducted using unripe, green bell pepper pericarp (Capsicum annuum L.).First, the incorporation of l-leucine into IBMP could be verified.As expected by the inverse isotope effect in GC a new peak eluting just before endogenous IBMP appeared in samples incubated with lleucine-d 10 .Based on mass spectral data the incorporation of nine deuterium atoms of l-leucine-d 10 (55) into IBMP-d 9 (56)  could be confirmed (Scheme 11). [88]urthermore, feeding studies using deuterated α-ketoisocaproic acid (α-KIC-d 7 ) proved the relevance of branched chain keto acids (BCKAs) in the biosynthesis of MPs.In fact, feeding experiments with l-leucine-13 C 6 , 15 N,2,3,3,4,5,5,5,5',5',5'-d 10 (l-leucine- 13 C 6 , 15 N, d 10 ) revealed that the 15 N-label is mostly lost during IBMP-biosynthesis indicating that BCKAs are incorporated preferentially. [88]nce another nitrogen source and C 2 -unit are needed to form a pyrazine motif, further investigations were performed using 13 C-and 15 N-labeled compounds.However, coeluting isotopologues due to a weaker inverse isotope effect for 13 C-/ 15 Natoms [90] and high IBMP-levels made the detection of 13 C-/ 15 Nincorporation impossible.That is why mixed labeling studies (co-feeding experiments) were performed by feeding a known deuterated precursor and the potential 13 C/ 15 N-labeled compound simultaneously. 13C/ 15 N-incorporations were detected by the biosynthetic product of both precursors.As the inverse isotope effect is enhanced by the incorporation of deuterium atoms, labeled and unlabeled isotopologues can be separated by GC × GC which in turn allows the detection of 13 C/ 15 Nincorporations.As displayed in Figure 9  Co-feeding experiments confirmed an incorporation of one nitrogen atom of glutamine and a C 1 -unit of glycine and glyoxylic acid.Consequently, it could be shown that glycine and glyoxylic acid are first metabolized to a C 1 -intermediate, which is then incorporated into the pyrazine heterocycle of IBMP (Figure 10). [88]he aim of the following study was thus to elucidate the underlying metabolic pathway of the metabolization of glycine and glyoxylic acid to a C 1 -intermediate.As shown in Scheme 12, glycine and glyoxylate are biochemically linked to the C 1metabolism via photorespiration.Photorespiration is often considered a wasteful and inefficient process, as it lowers the efficiency of photosynthesis.The main objective of the photorespiratory cycle might be the detoxification of 2-phosphoglycolate (2-PG), which is formed due to the fixation of O 2 catalyzed by the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO).During this detoxification, 2-PG is dephosphorylated to glycolate, which in turn is oxidized to glyoxylate.Glyoxylate is then aminated to glycine.One molecule of glycine undergoes a deamination and decarboxylation generating a C 1intermediate, which reacts with tetrahydrofolate (H 4 Folate) forming 5,10-methylene-tetrahydro-folate (CH 2 -H 4 Folate).CH 2 -H 4 Folate in turn reacts with another glycine molecule forming Scheme 9. Postulated biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP, 53) via leucinamide (AMPA, 52). [87]heme 10.Proposed incorporation of l-isoleucine (53) into (S)-sBMP (54).Retention of the stereochemistry of l-isoleucine during its incorporation. [56]heme 11.Incorporation of l-leucine-d 10 (55) generating IBMP-d 9 (56).Based on in vivo feeding experiments using green, unripe bell pepper pericarp (Capsicum annuum L.).l-serine. [91,92]l-Serine, as the final product of the metabolic interface of glycine and the C 1 -metabolism, was therefore considered a putative precursor for IBMP. [89]n vivo co-feeding experiments with unripe bell pepper pericarp (Capsicum annuum L.) did indeed prove that l-serine is incorporated into IBMP.Moreover, these results confirmed that l-serine is decarboxylated during the biosynthesis of IBMP and that it provides one nitrogen atom of the pyrazine ring. [89]ased on the feeding studies different biosynthetic pathways to IBMP were proposed (Scheme 13): I. l-Serine is decarboxylated to ethanolamine (57), which reacts with α-KIC and NH 3 forming IBHP.In plants, the decarboxylation of l-serine is known to be catalyzed by a serine decarboxylase (SDC).
A condensation of DAP and α-KIC results in IBHP.The nonproteinogenic amino acid DAP is a short-lived intermediate of the neurotoxin ß-N-oxalyl-α,ß-diaminopropionic acid (ODAP) found in grass pea (Lathyrus sativus L.). [95]V. l-Serine is oxidized to α-formyl-glycine (60), which reacts with α-KIC and NH 3 .Cyclization and decarboxylation lead to the formation of IBHP.α-Formyl-glycine is used for the chemical synthesis of cyclic endiamino peptides. [96]n addition, feeding experiments with l-serine strengthened the hypothesis of a metabolic link between IBMP-biosynthesis and photorespiration.Earlier feeding studies revealed that glutamine serves as a nitrogen source.Hence, NH 3 released by l-serine might be re-incorporated into IBMP via glutamine. [88]owever, three serine biosynthetic pathways coexist in plants.While the phosphorylated pathway is found in the plastids, the glycerate pathway occurs in the cytosol and the glycolate pathway (as a part of the photorespiratory cycle) in the mitochondrion.As displayed in Scheme 14 the glycerate pathway starts with a dephosphorylation of 3-phosphoglycerate (3- Scheme 14. Different biosynthetic pathways to serine in plants. [97]GA) to glycerate, which is oxidized to hydroxypyruvate.In the last step hydroxypyruvate forms serine.During the phosphorylated pathway 3-PGA is first oxidized to 3-phosphohydroxypyruvate, which is converted to 3-phosphoserine (PS).Finally, PS is dephosphorylated into serine.While the functional importance of the glycerate pathway is still unknown, the phosphorylated pathway turned out to be essential for the development of plant's embryos, male gametophytes as well as roots. [92,97]The involvement of these coexisting pathways to serine in the biosynthesis of MPs cannot be ruled out currently.

Translocation
In vivo feeding experiments using different plant organs and fruit tissues of bell pepper plants (Capsicum annuum L.) revealed that flowers, stems, and leaves biosynthesize IBMP de novo.Interestingly ripe, yellow bell pepper fruits showed no incorporation of l-leucine into IBMP. [61]Taking into account that the expression of important enzymes involved in MP-biosynthesis decreases during grapes ripening [87] it has been hypothesized that no de novo biosynthesis was detectable in ripe fruits due to the down-regulation of important enzymes. [61]Moreover, roots showed no incorporation of l-leucine or l-valine into IBMP or IPMP respectively, although they are especially rich in IPMP.That is why a transport of MP through the plant has been assumed to be responsible for the distribution of MPs throughout the plant. [61]Ps belong to the class of alkaloids, which are naturally occurring compounds containing at least one nitrogen atom, usually within a heterocyclic ring. [98]It is known that the site where a alkaloids are stored is not always their site of biosynthetic origin.The alkaloid nicotine (Figure 11, 61) for example is exclusively biosynthesized in the roots of tobacco plants (Nicotiana tabacum) but accumulates in the leaves. [99]In addition, senecionine (Figure 11, 62) and its N-oxide are biosynthesized in Senecio vulgaris L. roots and translocated through the shoot to finally accumulate in the inflorescences. [100]Hence, a transport of alkaloids and their intermediates through the plant via the vascular system of xylem and phloem is possible.A root-to-shoot transport of solutes into the leaves occurs usually unidirectional via the xylem. [101]In contrast, the transport of solutes via the phloem is bidirectional from source organs like leaves to sink organs such as fruits or seeds. [102] long-distance transport of MPs within the grapevine has been discussed controversially.On the one hand, experiments with a stable isotope labeled tracer (IBMP-d 2 ) indicated that IBMP is translocated from the leaves into the grapes. [65]Since the levels of added IBMP-d 2 were unnaturally high, the results of this work are difficult to interpret.On the other hand, grafting studies showed that there is no IBMP-transport through the shoot into the fruit. [103]However, IBMP-levels increased in the bunch stem (rachis) when they were grown on IBMP-rich rootstocks, so a transport through the shoot into the rachis has been suggested. [104]In addition, recent studies showed that IBHP-d 2 is found in all organs of grape vines after it is added to the roots, suggesting that the translocation of MPs to berry tissue may be inhibited, but not the translocation of HPs. [105] translocation of IBMP via the phloem was examined using the phloem sap of excised leaves of bell pepper plants.For phloem collection, methods like spontaneous exudation through simple incision, ethylenediaminetetraacetic acid(EDTA)facilitated phloem exudate collection or aphid stylectomy have been developed.EDTA-facilitated exudation is a commonly used method.For this purpose, tips of the petioles for instance are incubated in a solution containing EDTA, which serves as a chelating agent to prevent undesired phloem sealing. [106]nfortunately, the use of EDTA can lead to cell damages and thus false-positive results. [107]That is why control experiments need to be carried out.Samples obtained via the insect stylectomy method which requires phloem-feeding insects like aphids are less contaminated.For phloem collecting, the aphid's stylet which is inserted into the sieve tube while the aphid is feeding is cut off.Stylectomy is a less invasive, but more time-consuming and work-intensive phloem collecting technique. [108]he phloem sap of bell pepper plants was collected using the EDTA-facilitated exudation technique.The obtained phloem exudates were analyzed using HS-SPME-GC-ToF-MS.As no IBMP could be observed in the petiole phloem sap, it has been hypothesized that MPs themselves may not be phloem-mobile and need to be chemically modified first by for example a glycoconjugation. [61]A glycoconjugate of MP would be soluble in the phloem.In fact, glycosylated IBHP has already been considered to be a degradation product of IBMP. [35]Moreover, a translocation via the xylem has been considered possible, but has not yet been clearly demonstrated. [105]

Summary and Outlook
Since their first discovery in the 1960s, MPs have been of great research interest.Over the years, their occurrence in the plant kingdom has been extensively studied.IBMP turned out to be the most abundant MP in grapevines as well as in bell pepper plants.It is predominantly stored in the reproductive plant parts like fruits and flowers.In fact, the pericarp tissue of bell pepper fruits and the exocarp of grapes turned out to be the main site of IBMP-accumulation.Especially unripe fruit tissues are rich in IBMP. [60,61]Knowledge about the distribution of MPs and their site of storage may help to determine the biological function of MPs in plants.Distinct functions have been discussed in the literature, but none of them have been confirmed.Additionally, knowledge about the distribution of MPs may help to understand which viticultural practices reduce the MPcontent in grapes and wines.As elevated MP-concentrations in grapes can cause an unpleasant aroma in wine, knowledge about viticultural and environmental factors to reduce MPs in grapes is gaining importance. [109]For example, it could be shown that rachis are rich in IBMP, which is why it is recommended to exclude rachis tissue during winemaking. [110]oreover, cluster light exposure and higher temperatures turned out to affect IBMP-levels in grapes. [44,45,111]It is still unknown to this day whether light suppresses IBMP-biosynthesis or if it causes IBMP-degradation.Light-induced photodegradation and a thermal-induced degradation are discussed in the literature. [112]In addition, it could be shown that IBMPbiosynthesis is affected by biotic stresses.An up-and downregulation of IBMP-biosynthesis in response to stress caused by insects or bacteria has so far been observed in Coffea and Nicotiana benthamiana plants. [79,84]Furthermore, decreasing IBMP-levels and increasing IBHP-levels have also been observed during grape and bell pepper fruit ripening. [35,61]o figure out which processes are responsible for the decrease in MP-content at different ripening stages as well as in response to abiotic and biotic stresses, their biosynthetic pathway needs to be investigated.In vivo feeding experiments with stable isotope labeled compounds already gave an important insight into the biosynthesis of IBMP in plants.lleucine and α-KIC were identified as important IBMP-precursors.Moreover, it has been shown that glutamine serves as a nitrogen donor and that l-serine acts as a C 2 -building block and nitrogen source in the biosynthesis of IBMP. [88,89]Knowing the precursors of IBMP helps to elucidate its biosynthetic pathway and thus increases the knowledge of MP-biosynthesis significantly.In fact, different biosynthetic pathways for the formation of IBMP could be proposed based on the results of feeding studies. [89]However, for pathway delineation important intermediates and enzymes which participate in the biosynthesis of MPs need to be identified.
In addition, feeding experiments gave evidence for a metabolic link between IBMP-biosynthesis and photorespiration.This metabolic interface could be responsible for decreasing IBMP-levels in ripe fruits.A deficiency of the photorespiratory-derived l-serine during bell pepper ripening due to a decrease in activity of photorespiratory enzymes [113] may lead to a reduced IBMP-biosynthesis in ripe fruits.To prove this additional work is needed.In future work, it is necessary to examine whether photorespiration is the only l-serine-source in the IBMP-biosynthesis.
Finally, the genes of the MP biosynthesis pathway need to be identified.A powerful strategy to identify the genes involved in volatile synthesis is to exploit natural variation within the genus.Thus, populations of introgression lines (ILs) that contain defined segments of chromosomes from the wild relatives of tomatoes have been recently used to identify numerous volatile-associated quantitative trait loci (QTLs). [114]These QTLs, in turn, permitted identification of genes that define the synthetic pathway as well as points of regulation.A similar approach might lead to the identification of genes that play an essential role in MP production in fruits as a major step forward in understanding flavour quality.

Figure 10 .Scheme 13 .
Figure 10.Incorporation of l-leucine and α-KIC as well as glyoxylic acid, glycine, and glutamine into IBMP.Based on in vivo feeding experiments using green, unripe bell pepper pericarp (Capsicum annuum L.).The exact position of the 13 C-label in the pyrazine ring ( 13 C-5 or 13 C-6; here 13 C-5 is shown) cannot be determined at the present stage.